Green HPLC Method for Squalene Determination in Dietary Supplement

Abstract

The aim of this study was to develop a rapid and environmentally friendly method for determining squalene in dietary supplements. An Agilent 1200 chromatograph was used with Discovery F5 (SiO2 -PFP) and Discovery HS C18 (SiO2-C18) stationary phases, applying water–ethanol mixtures as mobile phases. To ensure environmental safety, toxic solvents (methanol, acetonitrile) were replaced with ethanol. Detection of squalene at 210 nm in ethanol-based mobile phases proved feasible. Comparison of the chromatographic phases showed that SiO2-PFP required 10–12% less ethanol than SiO2 -C18 to achieve equivalent retention, improving both efficiency and cost-effectiveness. A validated methodology for quantitative squalene determination was established. Optimal conditions were achieved with a Discovery F5 column (4.6×250 mm, 5 μm), thermostated at 40°C, and a mobile phase of 85% ethanol, with UV detection at 210 nm. The method met requirements of specificity, linearity, accuracy, and reproducibility. Analysis time per sample was only six minutes, and the use of ethanol ensured safety for both analysts and the environment. Application of the developed method revealed that the tested dietary supplement did not contain squalene. Instead, the undeclared presence of tocopheryl acetate was detected at 5.0 mass percent. This result underlines the importance of stricter regulatory oversight of dietary supplements on the Ukrainian market.